CORTECS 2.7 µm Columns
CONT ENT S
I.INT RODUC T ION
II.
G E T T ING STA RT E D
a. Column Connection
b. Column Installation
c. Minimizing Band Spread Volume
d. Column Equilibration
e. eCord™ Installation
f. Functional Tests for Benchmarking a New Column
g. VanGuard™ Cartridge Columns
III.
COLUMN US E
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
IV.
COLUMN C L EANING, R EG EN E RAT ION,
AND STO RAG E
a. Cleaning and Regeneration
b. Storage for Reversed-Phase and HILIC Columns
V.
eCO RD INT E L LIG ENT C HI P T EC HNOLOGY
a. Introduction
b. Installation
c. Column Use Information
V I.
A DDIT IONA L INFO RMAT ION
a. Tips for Maximizing Column Lifetime
b. Getting Started with CORTECS® HILIC Columns
c. Troubleshooting Questions
V II. C AUT IONA RY NOT E
I. INT RODUC T ION
Thank you for choosing a CORTECS Column. CORTECS 2.7 µm
Columns are based on the same solid-core particle technology
as CORTECS UPLC® 1.6 µm Columns, enabling seamless method
transfer between UPLC, UHPLC, and HPLC platforms. All CORTECS
Columns are manufactured in cGMP, ISO 9001:2000 certified
facilities. Strict quality procedures for the manufacture of the
solid-core particle, the bonded phase synthesis, and the column
packing result in a high quality and reproducible final product. Every
column is individually tested and a Performance Chromatogram
and Certificate of Batch Analysis are provided on the eCord
Intelligent Chip. The eCord is available on all 2.1 mm and
3.0 mm column dimensions.
CORTECS 2.7 µm Columns will exhibit maximum chromatographic
performance when used on a member of the ACQUITY UPLC® System
family since they were created and designed to operate with it.
II. G E T T ING STA RT E D
c. Minimizing Band Spread Volume
Each CORTECS 2.7 µm Column comes with a Certificate of
Analysis, which includes the bonded phase batch number and
the analytical test results for the unbonded and bonded particle.
T he included Performance Test Chromatogram summarizes
the performance of each individual column and provides batch
number, column serial number, USP plate count, USP tailing
factor, retention factor, and chromatographic test conditions.
These data should be recorded and stored for future reference.
When available, the information can be accessed via the
ACQUITY UPLC Console using the attached eCord.
Band spreading is the measurement of the system dispersion,
which impacts the chromatographic performance. Internal tubing
diameter and fluidic connections can significantly impact system
band spreading and chromatographic performance. Larger
tubing diameters cause excessive peak broadening and reduced
sensitivity (Figure 1).
0.005 inches
0.020 inches
0.040 inches
a. Column Connection
CORTECS 2.7 µm Columns are designed to operate on any HPLC,
UHPLC, or UPLC system. Due to the absence of an industry
standard, be aware that the type of fittings and connection on
each system will vary by manufacturer. All tubing connections
must be reseated to match the new column when it is installed.
If the style of the column end fitting does not properly match the
system connections, the chromatographic performance of your
new column will be negatively impacted, or leaking can occur.
b. Column Installation
Note. The flow rates given in the procedure below are described for
a 2.1 mm I.D. column. Adjust the flow rate up or down based on the
column pressure and system limits.
1. Purge the pumping system of any buffer-containing mobile
phases using 100% HPLC-grade water.
2. Purge the pumping system to 100% organic mobile phase
(methanol or acetonitrile).
3. Connect the inlet of the column to the chromatographic system.
4. Flush the column with 100% organic mobile phase (methanol
or acetonitrile) by setting the pump flow rate to 0.1 mL/min.
Increase the pump flow rate to 0.5 mL/min over 5 minutes.
5. Once the mobile phase is flowing from the column outlet, stop
the flow.
6. Attach the column outlet to the detector. This prevents air
from entering the detector flow cell.
7. Increase the flow rate as described in step 4.
Diluted/distorted sample band
Figure 1. Impact of tubing diameter on band spread.
d. Column Equilibration
CORTECS 2.7 µm Columns are shipped in 100% acetonitrile. It is
important to ensure mobile phase compatibility before changing
to a different mobile phase system. To avoid precipitating mobile
phase buffers within the column or system, flush the column with
five column volumes of a water/organic solvent mixture, using
the same, or lower, solvent content as in the desired buffered
mobile phase (for example, flush the column and system with 60%
methanol in water prior to introducing 60% methanol/40% buffer
mobile phase).
Note. If mobile phase additives (i.e., ion-pairing reagents) are
present in low concentrations (<0.2% v/v), 100 to 200 column
volumes may be required for complete equilibration. In addition,
mobile phases that contain formate (i.e., ammonium formate,
formic acid) may require extended equilibration times.
For reversed-phase separations, equilibrate the column with a
minimum of 10 column volumes of the mobile phase to be used
(refer to Table 1 for a list of column volumes). The column may be
considered fully equilibrated once a constant backpressure and a
stable detector baseline is achieved.
8. Monitor until a steady backpressure and baseline have
been achieved.
CORT ECS 2.7 µm Columns
2
Table 1. Empty Column Volumes (mL)
I.D.
Column length (mm)
1.0 mm
2.1 mm
30
0.024
0.10
50
0.039
75
0.059
100
0.079
150
0.12
3.0 mm 4.6 mm
0.21
0.50
0.17
0.35
0.83
0.26
0.53
1.25
0.35
0.71
1.66
0.53
1.07
2.49
For CORTECS HILIC Columns, flush with 50 column volumes of
50:50 acetonitrile/water with 10 mM final buffer concentration.
Prior to the first injection, equilibrate with 20 column volumes
of initial mobile phase conditions. If gradient conditions are
used, equilibrate with 8–10 column volumes between injections.
Failure to appropriately equilibrate the column could results in
drifting retention times. See “Getting Started with CORTECS HILIC
Columns” under “Additional Information”.
e. eCord Installation
eCord technology represents a significant advancement in column
usage tracking management when connected to an ACQUITY UPLC
System. The eCord Intelligent Chip can be read by connecting the
yellow fob to the reader/writer located on the right hand side of
the ACQUITY UPLC column heater module. Embedded information
such as the column manufacturing QC data and Certificates of
Analysis may be accessed via the ACQUITY UPLC console. The
eCord is available on all 2.1 mm and 3.0 mm column dimensions.
f. Functional Tests for Benchmarking a New Column
Waters recommends performing a benchmarking test upon receipt
of your column and throughout lifetime usage. By using a standard
mix, such as the appropriate QC Reference Material, you can:
■■
Verify the performance of your column upon receipt.
■■
Monitor the health of your column and system over time.
■■
Troubleshoot separation difficulties that arise.
1. Run an initial benchmark test to monitor key performance
criteria such as retention time, peak area, peak tailing,
resolution, response, system pressure, etc.
2. Repeat test daily to track column and system performance
over time.
For more information on benchmarking performance, visit
www.waters.com/QCRM
Note. If an analyte mixture found in the Performance Test
Chromatogram is used to benchmark, the isocratic efficiencies
measured in your laboratory may be less than those given on
the Waters Performance Test Chromatogram. This is normal
and expected. The Waters isocratic column testing systems
have been modified in order to achieve extremely low system
dispersion. This presents a more challenging test of how well
the column was packed. This also guarantees the highest
quality packed column. These special testing systems have
been modified to such an extent that they are not commercially
viable and have limited method flexibility other than isocratic
column testing.
3. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.
4. Repeat the test periodically to track column performance
over time. Slight variations may be obtained on different
LC systems due to the quality of the connections, operating
environment, system electronics, reagent quality, column
condition, and operator technique.
g. VanGuard Cartridge Columns
VanGuard Cartridge Columns are a universal solution to extend all
HPLC, UPLC and UHPLC CORTECS column lifetimes. The chemistries
offered for VanGuard Cartridge Columns are identical to the available
CORTECS chemistries. The reusable holder is designed with an
adjustable ferrule so that it can be attached to any column inlet.
VanGuard Cartridge Columns come in two sizes, 2.1 mm I.D. for
columns I.D.’s 3.0. mm or less, and 3.9 mm I.D. for column I.D.’s 3.9 mm
or greater.
The Neutrals QC Reference Material (p/n 186006360) or HILIC QC
Reference Material (p/n 186007226) are appropriate mixtures with
which to benchmark your CORTECS Column and system. Other QC
reference materials are available depending on the application and
detection mode.
CORT ECS 2.7 µm Columns
3
VanGuard Cartridge Column Installation Instructions
VanGuard Cartridge Columns are recommended to provided superior column protection for all HPLC and UHPLC columns up to
4.6 mm inner diameter.
VanGuard Cartridge Columns require the VanGuard Cartridge Holder (p/n 186007949) for proper assembly.
Cartridge Holder Parts list
Two Piece Ferrule
Holder Fitting
Protective Plug
Tools required
/ " wrench
/ " wrench
3 8
/ " wrench
5 16
7 16
O-Ring
Compression Screw
ATTENTION: READ AND UNDERSTAND ALL ASSEMBLY
INSTRUCTIONS PRIOR TO USE.
1. Remove protective o-ring.
Keep assembly upright to
keep ferrule from falling off.
DO NOT remove the protective
plug from the holder assembly
until Step 4. T he plug is used to
seat the tubing securely prior to
final assembly.
In order to ensure void-free
and leak-free connections, the
VanGuard Cartridge Holder is
shipped with the ferrule NOT
permanently attached. Care must
be taken when assembling the
holder to the column.
Transition Tube Assembly
Two Piece Ferrule
2. T hread the holder assembly
finger tight into the
column inlet.
3. With holder on top, tighten holder
to the column inlet using wrenches
(1/4 turn after finger tight).
Hold assembly securely
to prevent the holder
from slipping from
the column inlet.
Ensure that the
holder is firmly
seated into the
column inlet before
setting the ferrule.
HPLC column
⁄ " wrench
5 16
4. Remove plastic plug.
5. Insert guard cartridge
into holder.
Holder housing should
rotate freely until
cartridge is tightened.
6. Tighten guard cartridge.
⁄ " wrench
7 16
⁄ " wrench
38
Guard cartridge
Guard Cartridge Replacement Instructions
1. Loosen guard cartridge
and remove from holder.
2. Insert new guard cartridge
into holder.
The guard cartridge can be replaced
without removing the holder from
the column inlet.
3. Tighten guard cartridge.
⁄ " wrench
7 16
⁄ " wrench
7 16
⁄ " wrench
38
⁄ " wrench
38
CORT ECS 2.7 µm Columns
4
III. COLUMN US E
3. If the sample is not prepared in the mobile phase, ensure that
the sample, solvent, and mobile phases are miscible in order to
avoid sample precipitation or buffer precipitation.
To ensure the continued high performance of CORTECS 2.7 µm
Columns, follow these guidelines:
a. Sample Preparation
1. Sample impurities and/or particulates often contribute
to column contamination. One option to avoid column
contamination is to use Oasis ® or Sep-Pak ® solid-phase
extraction (SPE) devices. To select the appropriate sorbent for
a specific sample type, visit www.waters.com/sampleprep
2. It is preferable to prepare the sample in the initial mobile
phase conditions or a weaker solvent for the best peak shape
and sensitivity.
4. Filter sample with a 0.2 µm membrane to remove particulates.
If the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, methanol, etc.) ensure that the
membrane material is compatible with the solvents in use.
Alternatively, centrifuge the sample for 20 minutes at
8,000 RPM, followed by the transfer of the supernatant
to an appropriate vial.
5. For hydrophilic-interaction chromatography (HILIC) separations,
the samples must be prepared in a high percentage of organic
solvent (e.g., 95% acetonitrile). See “Getting Started with
CORTECS HILIC Columns” under “Additional Information”.
Table 2: CORTECS Chemistry Characteristics
Name of
column
Ligand type
Surface charge
modification
End cap
style
Carbon
load
Ligand
density
pH
limits
Temp.
limits
C18
Trifunctional C18
None
Proprietary
6.6%
2.7 µmol/m2
2–8
45 °C
5.7%
2.4 µmol/m
2
C18 +
Trifunctional C18
+
Proprietary
2–8
45 °C
Proprietary
4.5%
3.4 µmol/m
2
C8
Trifunctional C 8
None
2–8
45 °C
None
Proprietary
4.7%
1.6 µmol/m
2
T3
Trifunctional C18
2–8
45 °C
Shield RP18
Monofunctional
Embedded Polar C18
None
Proprietary
6.4%
3.2 µmol/m2
2–8
45 °C
Phenyl
Trifunctional phenyl
None
Proprietary
5.9%
3.2 µmol/m2
2–8
45 °C
None
None
None
Unbonded
n/a
1–5
45 °C
HILIC
Table 3. Mobile Phase Buffer Recommendations for CORTECS 2.7 µm Columns
Volatility
(±1 pH unit)
Used for
mass spec
0.30
Volatile
Yes
Ion pair additive, can suppress MS signal, used in
the 0.02–0.10% range.
Acetic Acid
4.76
Volatile
Yes
Maximum buffering obtained when used with
ammonium acetate salt. Used in 0.1–1.0% range.
Formic Acid
3.75
Volatile
Yes
Maximum buffering obtained when used with
ammonium formate salt. Used in 0.1–1.0% range.
Acetate
(NH4 CH2COOH)
4.76
3.76–5.76
Volatile
Yes
Used in the 1–10 mM range. Note that sodium or
potassium salts are not volatile.
Formate
(NH4 COOH)
3.75
2.75–4.75
Volatile
Yes
Used in the 1–10 mM range. Note that sodium or
potassium salts are not volatile.
Phosphate 1
2.15
1.15–3.15
Non-volatile
No
Traditional low pH buffer, good UV transparency.
Phosphate 2
7.20
6.20–8.20
Non-volatile
No
Above pH 7, reduce temperature/concentration
and use a guard column to maximize lifetime.
Additive/Buffer
pK a
TFA
Buffer range
Comments
CORT ECS 2.7 µm Columns
5
b. pH Range
Column lifetime will vary depending on the combination of
temperature, mobile phase pH, and type of buffer or additive
used. Table 3 lists the recommended buffers and additives for
CORTECS Columns.
Note. Working in combinations of extreme pH, temperature, and
pressure may result in reduced column lifetime.
solvent to remove the non-polar contaminant(s), taking care not to
precipitate any buffered mobile phase components. If this flushing
procedure does not solve the problem, purge the column with the
following cleaning and regeneration procedures.
1. Use a cleaning routine that matches the properties of the
samples and stationary phase type (reversed-phase, normalphase, or HILIC) to help solubilize the suspected contaminate.
2. Flush with 20 column volumes of solvent at 45 °C.
c. Solvents
To maintain maximum column performance, use high quality HPLC
or MS-grade solvents. Filter all aqueous buffers prior to use through
a 0.2 µm filter. Solvents containing suspended particulate materials
will generally clog the outside surface of the inlet of the column.
This may result in higher backpressure or distorted peak shape.
d. Pressure
CORTECS 2.7 µm Columns are compatible with HPLC, UHPLC,
and UPLC pressures. Table 4 provides the maximum operation
pressure for each column dimension.
Table 4. Maximum Operation Pressure
Column I.D.
Maximum operating pressure
2.1 mm
15,000 PSI [1034 bar]
3.0 mm
15,000 PSI [1034 bar]
4.6 mm
9,000 PSI [620 bar]
e. Temperature
CORTECS 2.7 µm Columns can be used at temperatures up to 45 °C
to enhance selectivity, reduce solvent viscosity, and increase
mass transfer rates.
IV. COLUMN C L EANING, R EG EN E RAT ION,
AND STO RAG E
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shouldering peaks, shifts in
retention, change in resolution, or increasing backpressure may
indicate contamination of the column. Flush with a neat organic
3. Return to the initial mobile phase conditions by reversing
the sequence.
4. If using a reversed-phase column, purge the column with a
sequence of progressively more non-polar solvents (i.e., waterto-methanol-to-tetrahydrofuran-to-methylene chloride).
5. If using a HILIC column, purge the column with a sequence of
progressively more polar-organic solvents (i.e., acetonitrileto-acetonitrile/methanol-to-acetonitrile/water-to-water).
6. If column performance has not improved after regeneration/
cleaning procedures, contact your local Waters representative
for additional support.
b. Storage after Reversed-Phase and HILIC Use
For periods longer than four days, store the column in 100%
acetonitrile. For separations utilizing elevated temperature, store
immediately after use in 100% acetonitrile. Do not store columns
in buffered eluents. If the mobile phase contained a buffer salt, flush
the column with 10 column volumes of HPLC-grade water (see Table
1 for column volume information) followed by 10 column volumes
of acetonitrile. Failure to perform this intermediate step could
result in precipitation of the buffer salt in the column when 100%
acetonitrile is introduced. Completely seal the column to avoid
solvent evaporation and drying out of the chromatographic bed.
Note. If a column has been run with a formate-containing mobile
phase (e.g., ammonium formate, formic acid, etc.) and is purged
with 100% acetonitrile, slightly longer equilibration times may be
necessary when the column is re-installed and re-wetted with that
same formate-containing mobile phase.
CORT ECS 2.7 µm Columns
6
V. eCO RD INT E L LIG ENT C HI P T EC HNOLOGY
c. Column Use Information
a. Introduction
The eCord intelligent chip provides the user with specific column
information as well as column use data including; chemistry
type, column dimension, serial number, and part number. The
overall column use information includes; total number of samples
injected, total number of injections as well as the maximum
pressure, and temperature that the column has been exposed to.
Additionally, detailed column history includes the sample set
start date, user name, and system name.
The eCord intelligent chip provides a paperless tracking history
of the column’s performance and usage throughout its lifetime.
The eCord is permanently attached to the column body via a
tether that cannot be removed. This ensures that the history of the
column is always accessible to the user of that column. The eCord
intelligent chip is available on CORTECS Columns with 2.1 mm
and 3.0 mm inner dimensions.
eCord–
intelligent chip
V I. A DDIT IONA L INFO RMAT ION
a. Tips for Maximizing Column Lifetime
Figure 5. eCord intelligent chip identification.
At the time of manufacture, the Performance Test Chromatogram,
analytical data for the particles, and Certificate of Batch Analysis is
downloaded onto the eCord. This information may then be accessed
via the ACQUITY UPLC console once the column is installed.
b. Installation of the eCord
The eCord device can be read by connecting the yellow fob to the
heater compartment receptacle located on the right hand side
of the ACQUITY UPLC Column heater module. Once the eCord
is connected, column identification, and overall column usage
information can be accessed.
eCord fob
Figure 6. Installing the eCord intelligent chip.
1. To maximize column lifetime, pay close attention to:
■■ Water quality (including water purification systems)
■■ Solvent quality
■■ Mobile phase preparation, storage, and age
■■ Sample, buffer, and mobile phase solubility
■■ Sample quality and preparation
2. When problems arise, systematically troubleshoot potential
cause one variable at a time in a systematic fashion.
3. Always remember to:
■■ Use an ACQUITY UPLC Column In-Line Filter Kit
(p/n 205000343) or a suitable Vanguard
Cartridge Column.
■■ Discourage bacterial growth by minimizing the use of
100% aqueous mobile phases where possible.
■■ Reduce the chances of mobile phase contamination or
degradation, prepare enough mobile phase to last for
3–4 days. Alternatively, store excess bulk quantities in a
refrigerated environment.
■■ Discard and re-prepare aqueous mobile phase every
24–48 hours (if 100% aqueous mobile phase is required).
■■ Add 5–10% organic modifier to aqueous buffer to minimize
bacterial growth (adjust gradient profile as necessary).
■■ Filter aqueous portions of mobile phase through a
0.2 µm filter.
■■ Routinely maintain your water purification system to
ensure it is functioning properly.
■■ Only use ultra-pure water (18 Ω-cm) and highest quality
solvent possible.
■■ Consider sample preparation (e.g., SPE, filtration,
centrifugation, etc.) when possible.
CORT ECS 2.7 µm Columns
7
4. Avoid when possible:
■■ 100% aqueous mobile phases.
■■ HPLC-grade bottled water.
■■ “Topping off” your mobile phases.
■■ Using phosphate salt buffer in combination with
high acetonitrile concentrations (e.g., >70%)
due to precipitation.
b. Getting Started with CORTECS HILIC Columns
Note. CORTECS HILIC Columns are designed to retain very polar
bases. Acidic, neutral, and non-polar compounds will have
limited retention.
Mobile Phase Considerations:
1. Always maintain at least 3% polar solvent, such as water,
in the mobile phase, or gradient. T his ensures that the
CORTECS HILIC particle is always hydrated.
2. Maintain at least 40% organic solvent (e.g., acetonitrile)
in your mobile phase or gradient.
3. Avoid phosphate salt buffers to avoid precipitation in high
organic content mobile phases used for HILIC separations.
Phosphoric acid is acceptable.
4. Buffered mobile phases such as ammonium formate or
ammonium acetate will produce more reproducible results
compared to unbuffered additives such as formic acid or acetic
acid. For best peak shape, maintain a buffer concentration
of 10 mM.
5. If using an ACQUITY UPLC System, the weak needle wash
solvent should closely match the percent organic solvent
present in the initial mobile phase conditions, otherwise,
analyte peak shape distortion can occur.
Injection Solvent Considerations:
1. Whenever possible, injection solvents should contain 95%
acetonitrile with the polar solvent (i.e., water, methanol,
isopropanol) component to be no more than 25% of the total
volume. A generic injection solvent is 75:25 acetonitrile/
methanol. T his is a good compromise between analyte
solubility and peak shape.
2. Avoid water and dimethyl sulfoxide (DMSO) in the injection
solvents. These solvents will produce very poor peak shapes.
In HILIC, it is important to remember that water is the
strongest eluting solvent. Therefore, it must be eliminated or
minimized in the injection solvent. Exchange water or DMSO
with acetonitrile by using reversed-phase SPE. If this is not
possible, dilute the water or DMSO with organic solvent.
c. Troubleshooting Questions
1. Are you using 100% aqueous mobile phases?
2. W hat is the age of the mobile phase?
3. Is the mobile phase filtered through a 0.2 µm membrane?
4. Was the mobile phase prepared fresh or topped off?
5. Is the water source of adequate quality?
6. W hen was the last time the water system was serviced or
was the bottle of water unopened?
7. Is bacterial growth a possibility (pH 7 phosphate buffer is
susceptible to bacterial growth within 24 hours)?
8. If a neat standard is prepared in the initial mobile phase
conditions and injected, are the problems still observed?
9. If the sample is filtered/purified (i.e., SPE, filtration… etc.) is
the problem still observed?
10. Has the quality of the samples changed over time?
V II. C AUT IONA RY NOT E
Some products may be hazardous during and after use and are
to be used by professional laboratory personnel trained in the
competent handling of such materials. The responsibility for the
safe use of products rests entirely with the purchaser and user.
The safety data sheets (SDS) for these products are available at
www.waters.com /SDS.
Waters, T he Science of W hat’s Possible, CORTECS, UPLC, ACQUITY UPLC, Alliance, Oasis, and Sep-Pak are registered trademarks of Waters
Corporation. eCord and VanGuard are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2016 Waters Corporation. Produced in the U.S.A. September 2016 720005024EN IH-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
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